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The Journal of Maternal-Fetal and Neonatal Medicine, 2013; 26(1): 75–78© 2013 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2012.728646

Objective: To describe associations among maternal/gesta-tional/neonatal characteristics and midpregnancy amniotic fluid concentrations of the main angiogenic markers vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Methods: In a cohort of 206 normal full-term pregnan-cies, midpregnancy amniotic fluid VEGF and PlGF reference values were recorded. Possible associations among the above concentrations and various parameters, such as maternal age and body mass index, race, parity, smoking, gestational age, delivery mode, birth-weight and fetal gender were investigated. Results: Midpregnancy amniotic fluid concentrations of both VEGF and PlGF increased with increasing gestational age (r = 0.173, p = 0.013 and r = 0.255, p < 0.001, respectively), whereas PlGF concentrations positively correlated with birth-weight (r = 0.154, p = 0.027). The effect of the other above-mentioned parameters on VEGF and PlGF concentrations was not signifi-cant. Conclusions: In normal pregnancies, midgestation amniotic fluid VEGF and PlGF concentrations positively correlate with gestational age. Furthermore, midgestation amniotic fluid PlGF concentrations may be a predictor of neonatal birth weight.

Keywords: VEGF, PlGF, angiogenic factors, pregnancy, amniotic fluid

IntroductionAngiogenic factors play a crucial role in the evolution of a normal pregnancy. In this respect, abnormal patterns of angiogenic markers have been linked to pregnancy complications, accounting for significant maternal and fetal morbidity [1,2]. Thus, women with preeclampsia, characterized by maternal hypertension and proteinuria usually after the 20th week of gestation [3], present with an abnormal angiogenesis profile, marked by elevated levels of antiangiogenic proteins and reduced concentrations of the main proangiogenic factors, free vascular endothelial growth factor (VEGF) and free placental growth factor (PlGF), both in serum and amniotic fluid, well before clinical disease onset [2,4,5]. Furthermore, several altered patterns of angiogenic markers have been reported to possibly contribute to restricted fetal growth [2,6], spontaneous preterm birth [7] and fetal death [8].

The angiogenic factors VEGF and PlGF are abundantly expressed by the placenta and several fetal tissues [9], and play key roles in the regulation of effective vasculogenesis, angiogen-esis and placental development [10]. Furthermore, both VEGF and PlGF are capable of inducing proliferation, migration and activation of endothelial cells, thus contributing to induction of vascular permeability and maintenance of the integrity of newly formed blood capillaries [11,12]. Failure to achieve the above adaptations may result in reduced feto-placental perfusion, char-acterizing disease states such as intrauterine growth restriction (IUGR) [6] and fetal death [8].

In both normal and pathological pregnancies, information is scarce regarding the associations between clinical pregnancy characteristics (including risk factors for adverse pregnancy outcomes) and maternal/amniotic fluid angiogenic profile. Pubmed/Medline was searched by use of the mesh terms “preg-nancy characteristics”, “gestational characteristics”, “maternal characteristics”, “angiogenic factors”, “angiogenic markers” and “neonates” in various combinations. The reference lists of the gathered reports were manually searched for additional studies. The search retrieved only a few studies, which have examined associations among maternal concentrations of angiogenic markers and maternal, gestational, as well as neonatal charac-teristics [13–15]. Furthermore, to the best of our knowledge, correlations of maternal, pregnancy and neonatal variables with second trimester amniotic fluid VEGF and PlGF values, have not been, up to the present, extensively investigated [16]. However, a deeper understanding of these relationships may provide new insights into the underlying mechanisms of adverse pregnancy outcomes and may improve control of confounding in studies testing the screening/diagnostic potential of angio-genic markers.

Therefore, the present study aimed to provide second trimester amniotic fluid VEGF and PlGF reference values, and also describe the potential relationship among the above concentrations and a variety of maternal characteristics (demographic/anthropometric/lifestyle), gestational parameters, as well as neonatal features. The study group comprised of normal full-term pregnancies, in order to exclude the most prevalent complications that are associated with altered angiogenic patterns, thus, permitting a clearer interpretation of the results.

Midtrimester amniotic fluid concentrations of angiogenic factors in relation to maternal, gestational and neonatal characteristics in normal pregnancies

Thalis Papapostolou1, Despina D. Briana1, Maria Boutsikou1, Christos Iavazzo1, Karl-Phillip Puchner1, Dimitrios Gourgiotis2, Antonios Marmarinos2 & Ariadne Malamitsi-Puchner1

1Second Department of Obstetrics and Gynecology, Athens University Medical School, Athens, Greece and 2Laboratory of Clinical Biochemistry-Molecular Diagnostics, 2nd Department of Pediatrics, Athens University Medical School, Athens, Greece

Correspondence: Ariadne Malamitsi-Puchner, MD, Second Department of Obstetrics and Gynecology, Athens University Medical School, 19, Soultani Street, 10682 Athens, Greece. Tel: +30 6944443815, Fax: + 30 2107233330. E-mail: amalpu@aretaieio.uoa.gr, amalpu@tellas.gr

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MethodsThe Ethics Committee of our teaching hospital approved the study protocol. Written informed consent was obtained by all participants.

Two hundred and twenty-seven Greek women with singleton pregnancies, who underwent amniocentesis at a median (range) gestational age of 17.56 (14.72–25) for standard genetic indica-tions (mostly because of maternal age or family history of genetic disorders) during a period of 16 months, were prospectively recruited.

Eligible women had normal pregnancies, which were defined as (a) term delivery (>37 gestational weeks) (b) delivery of an appropriate-for-gestational-age neonate (birth weights ranged between the 11th and 90th customized centile, controlling for maternal height, booking weight, ethnic group, parity, gestational age, birth weight and neonatal gender) [17] (c) absence of hyper-tensive disorders, diabetes mellitus and any other obstetrical or medical complications, including active vaginal bleeding, chronic renal, liver or heart diseases, chronic respiratory insufficiency, thyroid and immunological diseases and congenital or acquired thrombophilic disorders. Hypertensive disorders, that were ascer-tained through review of prenatal and labour records, included diagnosis of chronic hypertension before pregnancy, gestational hypertension (minimal criteria: new-onset hypertension after 20 weeks of gestation with systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg without any systemic features of preeclampsia and normal blood pressure levels within 3 months postpartum), or preeclampsia (defined as blood pres-sure augmentation after 20 weeks of gestation to >140/90 mmHg on at least two occasions 6 h apart in a previously normotensive woman, combined with proteinuria defined as protein dip stick ≥1+ on ≥2 midstream urine samples 6 hours apart or a 24-h urine excretion of ≥0.3 g protein in the absence of urinary infection) [18]. Furthermore, multiple pregnancies and pregnancies with abnormal karyotype or major fetal anomalies were excluded from the study.

Maternal demographic, anthropometric and lifestyle charac-teristics, as well as a detailed obstetrical and personal medical history were obtained by interview at enrolment. Smoking was grouped as follows: never smoked, smoked but quit when preg-nancy confirmed, smoked less than 10 cigarettes/day before and during pregnancy, and smoked more than 10 cigarettes/day before and during pregnancy. Pre-pregnancy weight and maternal height were used to calculate pre-pregnancy body mass index (BMI), which was grouped according to the Centers for Disease Control guidelines into underweight, normal, overweight and obese [19]. The gestational age was calculated from the last menstruation and was confirmed by ultrasound measuring of the biparietal diameter and the femur length.

Ultrasound-guided transabdominal amniocentesis for stan-dard genetic indications was performed under sterile conditions. An average of 20 ml of amniotic fluid was obtained and none of the samples were contaminated by maternal blood. The amniotic fluid was separated in 3 parts. The biggest part (about 15 ml) was used for karyotype determination. The second part (about 2 ml) was transferred to sterile vacuum containers, in order to be tested by polymerase chain reaction (PCR) for Mycoplasma hominis and Chlamydia trachomatis and be cultured for standard aerobic and anaerobic bacteria. The third part (about 3 ml) was refriger-ated at +4ºC and centrifuged the next 6 h for 10 min. The superna-tant fluid was stored in polypropylene tubes at –80ºC.

The determination of amniotic fluid VEGF and PlGF concen-trations was performed by ELISA (Orgenium Laboratories,

07120, Vantaa, Finland and DRG Instruments, 35039, Marburg, Germany, respectively).

The minimum detectable concentrations, intra- and interassay coefficients of variation were <5 pg/ml, <6% and <10% for VEGF and <1.062 pg/ml, 2.83% and 4.10% for PlGF, respectively.

Statistical analysis

Data regarding both VEGF and PlGF were not normally distrib-uted (Kolmogorov–Smirnov test); thus non parametric proce-dures were used in the analysis. Mann–Whitney U test was applied to examine the effect of maternal age and weight, BMI, race, parity, smoking, gestational age, spontaneous rupture of membranes, delivery mode, birth weight and fetal gender on VEGF and PLGF levels, while Spearman’s correlation co-efficient was used to examine any relative positive or negative correla-tions. A p < 0.05 was considered statistically significant. SPSS 11.5 statistical package was used for the analysis.

ResultsTwenty one women were excluded, due to failure meeting the inclusion criteria. Thus, the study population comprised of 206 participants. The characteristics of the study population are shown in Table I.

Reference values (median [range]) were 126.56 (45.30–518.92) pg/mL and 18.81 (5.61–168.61) pg/mL for amniotic fluid VEGF and PlGF concentrations, respectively. Distribution of second trimester amniotic fluid VEGF and PlGF concentrations in our population are shown in Figures 1 and 2.

Amniotic fluid concentrations of both VEGF and PlGF increased with increasing gestational age (r = 0.173, p = 0.013 and r = 0.255, p < 0.001, respectively), at the time of amniocen-tesis. Furthermore, amniotic fluid PlGF concentrations positively correlated with birth-weight (r = 0.154, p = 0.027). The effect of maternal age and weight, BMI, race, parity, smoking, delivery mode and fetal gender on amniotic fluid VEGF and PlGF concen-trations was not significant.

DiscussionThe data of this study provide a useful set of VEGF and PlGF reference values in midpregnancy amniotic fluid. Furthermore, the present report investigates associations among established risk factors for adverse pregnancy outcomes and concentrations of second trimester amniotic fluid VEGF and PlGF in normal pregnancies.

More specifically, our findings indicate that second trimester amniotic fluid concentrations of both VEGF and PlGF increase with increasing gestational age. Studies investigating maternal circulating angiogenic markers at different gestational weeks have noted a rise in PlGF levels at midpregnancy [2,15]. Similarly, amniotic fluid PlGF concentrations were found to be significantly higher in midtrimester pregnancy than at term [16]. On the other hand, placental VEGF expression as well as amni-otic fluid VEGF concentrations reportedly decline as pregnancy advances [20]. Overall, both angiogenic factors increase between the first and second trimesters, consistent with an increased level of angiogenesis as the placenta develops [21].

Furthermore, our data suggest a positive correlation between amniotic fluid PlGF concentrations and neonatal birthweight. Some investigators have reported lower PlGF levels in women who did not experience preeclampsia, but delivered small-for-gestational-age infants [2,22]. However, in normotensive

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pregnancies, antiangiogenic maternal serum profile at delivery did not appear to be associated with neonatal weight percentile [13]. The authors speculate that maternal angiogenic alterations at the level observed in otherwise uncomplicated pregnancies may have little effect on perinatal health [13].

On the other hand, maternal and fetal PlGF levels are lower in IUGR, suggesting the formation of an antiangiogenic state [23]. In accordance, maternal and neonatal PlGF concentrations positively correlated with the infants’ customized centiles in both IUGR and normal pregnancies in a previous study from our group [24]. Most recent preliminary data also suggest that decreased concentrations of PlGF in maternal circulation may differentiate IUGR due to placental insufficiency from constitutionally small fetuses [25]. Nevertheless, our results possibly suggest that not only maternal, but also midpregnancy amniotic fluid PlGF levels may be a good indicator of neonatal birth weight.

Moreover, our data suggest that midpregnancy amniotic fluid VEGF and PlGF levels are independent of maternal BMI and age, parity, race, smoking, delivery mode and fetal gender.

Overall, studies that have examined the association between maternal pre-pregnancy BMI/weight and angiogenic markers have found inverse relations, although findings have not been consistent [13,15,26]. Given that high BMI is a risk factor for preeclampsia [27], the relationship among angiogenic markers, BMI and pregnancy outcome requires further investigation.

Maternal age and parity with respect to angiogenic markers have been examined in a few studies. Associations between maternal age and first-trimester levels of PlGF have been incon-sistent [28], whereas for levels at delivery a recent report demon-strated that in normotensive pregnancies, increasing maternal age is associated with a more antiangiogenic profile, including lower PlGF concentrations [13]. The authors suggest that a greater antiangiogenic profile with older maternal age may suggest a biological mechanism which mediates this preeclampsia risk factor [13]. Nevertheless, in line with our results, maternal age was not associated with maternal VEGF or PlGF concentrations at midpregnancy in a large recent study [15].

Nulliparity is the predominant independent risk factor for preeclampsia, but the mechanism of this association is unknown [29]. It has been suggested that increased secretion of circulating soluble fms-like tyrosine kinase (sFlt-1), an inhibitor of PlGF and VEGF in first versus second pregnancies, may in part account for the increased risk of preeclampsia among nulliparous women [14]. The authors speculate that the relative antiangiogenic profile

Table I. Maternal, gestational and neonatal characteristics of the study population.

Mean ± SD/median (range)Maternal age (years) 37 (21–50)Maternal weight (kg) 63.78 ± 11.05Maternal height (m) 1.66 ± 0.13BMI (kg/m2) 22.9 (16.0–41.2)Gestational age at amniocentesis (weeks) 17.56 (14.72–25)Gestational age at delivery (weeks) 38.53 (37.0–41.0)Birth weight (g) 3220.39 ± 301.8Customized centile 40 (11–90)BMI (categorical) N (%) Underweight 6 (2.9) Normal 143 (69.4) Overweight 47 (22.8) Obese 10 (4.9)Ethnicity N (%) Greek 199 (96.6) Other 7 (3.4)Parity N %) First 93 (45.1) Other 113 (54.9)Gender (N%) Male 112 (54.4) Female 94 (45.6)Mode of Delivery N (%) Vaginal 93 (45.1) Cesarean section 113 (54.9)Smoking N (%) Never smoked 141 (68.5) Ex-Smoker 47 (22.8) Smoking before/during pregnancy

(<10 cigarettes per day)5 (3.4)

Smoking before/during pregnancy (>10 cigarettes per day)

13 (6.3)

Figure 1. Distribution of second trimester amniotic fluid vascular endothelial growth factor (VEGF) concentrations.

Figure 2. Distribution of second trimester amniotic fluid placental growth factor (PlGF) concentrations.

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observed in nulliparous women may be responsible for the remarkable tendency of preeclampsia to occur in these women [14]. As far as maternal PlGF is concerned, lower concentrations have been demonstrated in nulliparous women, irrespective of gestational weeks at measurement [13,21,28], while Mijal et al did not observe a positive association between multiparity and maternal PlGF levels at midpregnancy [15].

Paradoxically, it has been noted that cigarette smoking reduces the risk of preeclampsia. This effect has been verified by a number of investigators [30] and it has been hypothesized that women who smoke during pregnancy may have a more favorable angiogenic profile than women who do not. Indeed PlGF levels measured anytime during pregnancy were higher among women who smoked during pregnancy compared with those who did not [15]. However, maternal smoking did not have any effect on amniotic fluid PlGF concentrations in the present study.

Finally, differences in angiogenic marker levels by race/ethnicity have been documented in a small number of reports [15].

Advantages of the present study include the prospective collec-tion of amniotic fluid samples in a clearly defined population, the use of an uncomplicated pregnancy group and the exploration of little-studied characteristics.

In conclusion, our findings suggest that in normal pregnancies, midpregnancy amniotic fluid VEGF and PlGF concentrations increase with increasing gestational age. Furthermore, second trimester amniotic fluid PlGF concentrations positively correlate with neonatal birth weight. Larger studies are needed to confirm these findings and their etiological implications, as well as further investigate their relevance for the short and long-term health of the mother and offspring.

Declaration of Interest: The authors report no declarations of interest.

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